mutagenize plasmid-encoded gene - (Apr/12/2010 )

I would like to knock-out a bacterial gene that is encoded on a large native plasmid by insertional mutagenesis using the targetron system. My question is, are there any additional difficulties associated with targeting a plasmid gene for knock-out, given that there are multiple copies of the plasmids. For example, if your selective marker gets inserted into one of the plasmid copies, is it still possible that some of the original plasmids carrying the native gene will be maintained in the host, despite plating with your antibiotic?

I've been looking for a reference where any plasmid gene was knocked-out using any technique - I'm sure there are hundreds - but as you might imagine, searching for "mutagenesis" and "plasmid" doesn't exactly produce these specific results. If anyone has some experience with this, or could point me towards some references, it would be greatly appreciated.

Thanks in advance.

-n0idbi0n-

n0idbi0n on Apr 12 2010, 02:15 PM said:

Hi there,

I would like to knock-out a bacterial gene that is encoded on a large native plasmid by insertional mutagenesis using the targetron system. My question is, are there any additional difficulties associated with targeting a plasmid gene for knock-out, given that there are multiple copies of the plasmids. For example, if your selective marker gets inserted into one of the plasmid copies, is it still possible that some of the original plasmids carrying the native gene will be maintained in the host, despite plating with your antibiotic?

I've been looking for a reference where any plasmid gene was knocked-out using any technique - I'm sure there are hundreds - but as you might imagine, searching for "mutagenesis" and "plasmid" doesn't exactly produce these specific results. If anyone has some experience with this, or could point me towards some references, it would be greatly appreciated.

Thanks in advance.

It is likely you will have native and mutant plasmids following mutagenesis. You may be able to increase the concentration of the antibiotic, depending on which one you are using, to select for higher numbers of mutant plasmids. The drawback of that, however, is you don't have a way to show that the increased antibiotic pressure doesn't have an effect somewhere else in the genome. A lot will depend on how many copies the plasmid actually is. Lower numbers will be easier to mutate, obviously. I can't point you in any particular direction for references.

-fishdoc-

fishdoc on Apr 12 2010, 03:45 PM said:

n0idbi0n on Apr 12 2010, 02:15 PM said:

Hi there,

I would like to knock-out a bacterial gene that is encoded on a large native plasmid by insertional mutagenesis using the targetron system. My question is, are there any additional difficulties associated with targeting a plasmid gene for knock-out, given that there are multiple copies of the plasmids. For example, if your selective marker gets inserted into one of the plasmid copies, is it still possible that some of the original plasmids carrying the native gene will be maintained in the host, despite plating with your antibiotic?

I've been looking for a reference where any plasmid gene was knocked-out using any technique - I'm sure there are hundreds - but as you might imagine, searching for "mutagenesis" and "plasmid" doesn't exactly produce these specific results. If anyone has some experience with this, or could point me towards some references, it would be greatly appreciated.

Thanks in advance.

It is likely you will have native and mutant plasmids following mutagenesis. You may be able to increase the concentration of the antibiotic, depending on which one you are using, to select for higher numbers of mutant plasmids. The drawback of that, however, is you don't have a way to show that the increased antibiotic pressure doesn't have an effect somewhere else in the genome. A lot will depend on how many copies the plasmid actually is. Lower numbers will be easier to mutate, obviously. I can't point you in any particular direction for references.

Thanks for the reply. We believe the plasmids are very low copy - only a few per cell. I would imagine that over generations the plasmids not carrying the selective marker would be randomly lost and after a few subcultures only the marked plasmid would remain, although I have no evidence for this.

-n0idbi0n-

n0idbi0n on Apr 12 2010, 03:16 PM said:

Thanks for the reply. We believe the plasmids are very low copy - only a few per cell. I would imagine that over generations the plasmids not carrying the selective marker would be randomly lost and after a few subcultures only the marked plasmid would remain, although I have no evidence for this.

We had a plasmid (about 6 kb) that was randomly mutated by signature-tagged mutagenesis. I'm not sure of it's copy number, but I figure between 20 and 40 copies per cell. Both copies we maintained quite well. I was only able to get rid of the the native plasmid by using 500x our normal concentration of antibiotic. With yours being much lower, I think you'd probably have a better shot.

Would it be possible to cure the plasmid, then reintroduce it with the mutation?

-fishdoc-

fishdoc on Apr 12 2010, 05:04 PM said:

n0idbi0n on Apr 12 2010, 03:16 PM said:

Thanks for the reply. We believe the plasmids are very low copy - only a few per cell. I would imagine that over generations the plasmids not carrying the selective marker would be randomly lost and after a few subcultures only the marked plasmid would remain, although I have no evidence for this.

We had a plasmid (about 6 kb) that was randomly mutated by signature-tagged mutagenesis. I'm not sure of it's copy number, but I figure between 20 and 40 copies per cell. Both copies we maintained quite well. I was only able to get rid of the the native plasmid by using 500x our normal concentration of antibiotic. With yours being much lower, I think you'd probably have a better shot.

Would it be possible to cure the plasmid, then reintroduce it with the mutation?

That's a thought, however the plasmid is quite large (~80-90 kb), and there are also other large plasmids carried by the same strain, so it would be difficult to cure only the specific plasmid of interest, as well as to isolate and work with a plasmid that large.